Cancer of the uterine cervix (CxCa) is the second most common malignancy in women worldwide and is caused by high-risk human papillomaviruses with HPV16 being the most prevalent type. In developed countries, conventional cytological screening programs have substantially reduced the incidence of this kind of cancer. These cytological screening programs, however, have some drawbacks.
The Papanicolaou test, frequently also referred to as Pap test, is a diagnostic method designed for the detection of premalignant and malignant lesion in the uterine cervix. For the Papanicolaou test, samples are obtained from the cervix and screened by light microscopy for changes in cell morphology indicating malignant or premalignant cells. Then, samples are classified depending on the severity of the observed lesions. However, diagnosis by cervical cytology is a subjective method, and the quality depends on the standards of the laboratory that provides the service. As such, lesion categorization is only moderately reproducible and of poor sensitivity compared to colposcopy (Baldwin, P., R. Laskey, and N. Coleman. 2003. Translational approaches to improving cervical screening. Nat Rev Cancer 3:217-26). Moreover, false positive results lead to a high number of patients that are being over-treated.
Within the last two decades a variety of new diagnostic tests for HPV were developed. These methods are based on the detection of viral, molecular and biochemical markers, such as HPV proteins, DNA and RNA.
The FDA-approved Hybrid Capture II Test System (HC2) (formerly Digene Corp., USA, now Qiagen, Germany) is considered the gold standard for HPV DNA testing in clinical practice, however, it shows several disadvantages: a) no genotyping is performed, instead HPV infection is solely attributed to a “low-risk” or “high-risk” group, b) multiple infections cannot be identified, c) it is less sensitive for HPV detection than PCR-based methods (Birner et al. 2001. Mod. Pathol. 14:702-709), and d) it is modestly specific for predicting of cervical precancer and cancer risk. Some of its non-specificity for clinical end points can be ascribed to cross-reactivity with non-carcinogenic HPV genotypes (Castle, P. E., D. Solomon, C. M. Wheeler, P. E. Gravitt, S. Wacholder, and M. Schiffman. 2008. Human papillomavirus genotype specificity of hybrid capture 2. J Clin Microbiol 46:2595-604). Moreover, it only allows for the assessment whether a subject is infected with HPV or not. The test does not allow for assessing the severity of a HPV infection. Thus, once HPV has been diagnosed, further examinations are required.
Several PCR-based methods were developed within the last years, allowing a more precise detection of HPV infection. The majority of these PCR systems use consensus or general primers that bind to highly conserved regions of the HPV genome, e.g. in the L1 region.
The amplified PCR products are then subjected to further analysis (e.g. sequencing, restriction fragment length polymorphism (RFLP) analysis or hybridization) in order to identify specific mucosal HPV genotypes. Longitudinal cohort studies have shown that combined Pap and HPV testing exhibit better sensitivity and predict better long-term protection (among women with normal results of both tests) against CIN3 than cytological testing alone (Bulkmans, N. W., J. Berkhof, L. Rozendaal, F. J. van Kemenade, A. J. Boeke, S. Bulk, F. J. Voorhorst, R. H. Verheijen, K. van Groningen, M. E. Boon, W. Ruiting a, M. van Ballegooijen, P. J. Snijders, and C. J. Meijer. 2007. Human papillomavirus DNA testing for the detection of cervical intraepithelial neoplasia grade 3 and cancer: 5-year follow-up of a randomised controlled implementation trial. Lancet 370:1764-72, Hoyer, H., C. Scheungraber, R. Kuehne-Heid, K. Teller, C. Greinke, S. Leistritz, B. Ludwig, M. Durst, and A. Schneider. 2005. Cumulative 5-year diagnoses of CIN2, CIN3 or cervical cancer after concurrent high-risk HPV and cytology testing in a primary screening setting. Int J Cancer 116:136-43.). However, the high sensitivity of HPV PCR tests leads also to the identification of clinically not relevant infections or regressing lesions. Therefore, the positive predictive value (PPV) for the presence of an advanced lesion or the development of cervical cancer after an individual high-risk HPV DNA positive result is low. The resulting high proportion of test-positive but disease-negative diagnoses cause over-treatment, additional costs and considerable anxiety for women concerned (International Agency for Research on Cancer. 2005. Cervix Cancer Screening. IARC Press, Lyon).
Unlike HPV DNA testing, RNA detection allows the identification and analysis of transcriptionally active viruses. A recent introduction of preservation media for cervical smears that, apart from DNA and cell morphology, also conserves RNA, enhanced the development of RNA detection methods. To date, two commercial HPV RNA detection assays have been introduced: i) PreTect HPV Proofer® from BIOMERIEUX® (formerly NorChip) that detects early full-length mRNA targeting E6 and E7 sequences (E6/E7) from hrHPV types 16, 18, 31, 33 and 45, and ii) the Aptima® HPV test, a broad spectrum E6/E7 full-length mRNA amplification method from Gen-Probe®. Limited data from these tests indicate that testing for full-length HPV E6/E7 mRNA rather than HPV DNA alone only slightly increases the PPV for the development of cervical cancer and its precursors, while at the same time, sensitivity and thus the negative predictive value (NPV) is reduced (Cuschieri, K. S., M. J. Whitley, and H. A. Cubie. 2004. Human papillomavirus type specific DNA and RNA persistence—implications for cervical disease progression and monitoring. J Med Virol 73:65-70). The main disadvantage of these technologies refers to the fact that they cannot predict disease due to only qualitative measurement of a single full-length viral oncogene transcript. Moreover, cervical smears can comprise different amounts of HPV-infected cells that cannot be controlled for by these technologies.
The development of cervical cancer is closely linked to the integration of the HPV genome into the chromosome of the host cells. In low-grade lesions, the majority of HPV genomes are present in an episomal state, whereas in high-grade lesions and carcinoma, the HPV genome can be integrated into the host genome. However, it has been demonstrated that not in all cases of cervical carcinoma the HPV genome is present in an integrated form (Vinokurova, S., N. Wentzensen, I. Kraus, R. Klaes, C. Driesch, P. Melsheimer, F. Kisseljov, M. Durst, A. Schneider, and M. von Knebel Doeberitz. 2008. Type-dependent integration frequency of human papillomavirus genomes in cervical lesions. Cancer Res 68:307-13.). Integration of the HPV16 genome into the host genome is only found in app. 60% of cervical cancer cases. Thus, diagnostic means which determine only the integration status of the HPV genome are not reliable for risk stratification.
Colposcopy allows for examining the uterine cervix and vagina. By this visual examination, many premalignant lesions and malignant lesions in these areas can be detected. Due to its high reliability, colposcopy is regarded to be the goldstandard for diagnosing cervical diseases. This diagnostic procedure, however, is cost- and time-intensive. Colposcopy requires highly trained personnel and often involves an invasive procedure (biopsy with subsequent histologic analysis). Consequently, colposcopy cannot be used in cervical cancer precursor screening programs.
The technical problem underlying the present invention may be seen as the provision of means and methods for efficiently and reliably differentiating between mild and severe forms of HPV16 infection without the drawbacks as referred to above. Also, means and methods are required for a reliable risk stratification of subjects not having the HPV genome integrated into the genome. The technical problem is solved by the embodiments characterized in the claims and herein below.